T cell clonal anergy, a nonresponsive state characterized by a critical reduction in the ability of the T cell to produce interleukin-2 (IL-2), is a proposed mechanism for peripheral and/or thymic tolerance. The biochemical basis for anergy is unknown. We have recently cloned the gamma isoform of multifunctional Ca2+/calmodulin-dependent protein kinase (CaM Kinase) from lymphocytes and have demonstrated its activation after T cell receptor (TCR) stimulation. Lymphocyte CaM kinase retains characteristic regulatory features previously demonstrated in neuronal isoforms, including autophosphorylation at Thr286, converting it to a partially Ca2+-independent species. Our preliminary studies indicate that CaM kinase negatively regulates IL-2 expression in Jurkat cells, suggesting that CaM kinase may play a role as the Ca2+ dependent effector molecule mediating anergy. Our overall objective in the proposal is to further substantiate a role for CaM kinase in anergy by addressing the following specific aims: (1) by means of coexpression of constitutive (versus inactive) CAM kinase and IL-2 reporter gene constructs, to document negative regulation of IL-2 transcription by CaM kinase; (2) by means of specific CaM kinase inhibitors, to demonstrate involvement of CaM kinase in an established model in which cloned T cells are rendered anergic by presentation of antigen on MHC transfected L cells; (3) by means of coexpression of constitutive CaM kinase, constitutive calcineurin, and IL-2 reporter gene constructs, to determine if the negative regulatory action of CaM kinase resides upstream, downstream, or at the level of calcineurin action; (4) to determine the effect of the costimulatory signal via the CD28 receptor on the Ca2+ signal, calmodulin availability, and the relative activation of CaM kinase versus calcineurin.